Broccoli type having curds with detached florets

ABSTRACT

The present invention includes broccoli plants with curds having detached florets and methods for obtaining such broccoli plants. The present invention also provides reagents that can be used in methods for obtaining such broccoli plants.

CROSS-REFERENCE TO RELATED CASES

This application is a continuation of U.S. application Ser. No.15/047,293, filed Feb. 18, 2016 (allowed), which is a continuation ofU.S. application Ser. No. 13/987,035, filed Jun. 27, 2013 (abandoned),which is a continuation of U.S. application Ser. No. 13/573,221, filedAug. 31, 2012 (now U.S. Pat. No. 9,220,208), which is a continuation ofU.S. application Ser. No. 13/194,557, filed Jul. 29, 2011 (now U.S. Pat.No. 8,304,615), which is a division of U.S. application Ser. No.11/602,276, filed Nov. 21, 2006, (now U.S. Pat. No. 8,026,416), whichclaims priority under 35 U.S.C. § 119(e) to provisional U.S. ApplicationNo. 60/739,803, filed Nov. 22, 2005, all of which are incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the field of plant breeding and thedevelopment of new plants. More specifically, to the development of anew and distinct broccoli type (Brassica oleracea L. var. italica)having curds with detached florets that are uniformly green, i.e., showno yellowing or discoloration around the edges.

Background of the Invention

The botanical family to which broccoli belongs is the Brassicaceae (orreferred to as Cruciferae), which is also known as the mustard family(or familiarly named as crucifers, as each flower has four distinctsepals and petals in the form of a cross). The Brassicaceae is a largefamily comprised of approximately 3,000 described species apportionedamong 350 to 380 genera. The precise number of genera will varydepending on the authority. The classification scheme for broccoli andindeed for all of the other brassica's is clear and straightforwarduntil one reaches the species level. At that point the addition ofnumerous subspecies or cultivar groups results in a rather complex andconfusing arrangement of the taxa in question. For example, thescientific name for broccoli, Brassica oleracea (L.), is also shared bycabbage, Chinese cabbage, cauliflower, collards, Brussels sprouts, kale,kohlrabi, to name a few. Despite the fact that all of the aforementionedtypes are sexually compatible, and are therefore referred to as B.oleracea, they are nevertheless separate entities.

The genus Brassica includes a number of notable vegetable crops otherthan Brassica oleracea (2n=18). The Brassica genus also includesvegetables or forage crops of many other genera. Most importantly inthis context are B. rapa (2n=20) or turnip and B. napus (2n=38) orswede. At the species level taxonomic names used for broccoli areBrassica oleracea L. convar botrytis (L) Alef: var. cymosa Duch,Brassica oleracea L. convar botrvtis (L) Alef. var. italica Plenck andBrassica oleracea L. var italica Plenck. Taxonomic experts havedifferent opinions for reasons mentioned before. The Germplasm ResourcesInformation Network (GRIN), of the United States Department ofAgriculture, uses Brassica oleracea L. var italica Plenck in the GRINTaxonomy for Plants to apply to broccoli. Broccoli is hereinafterreferred to as Brassica oleracea L. var italica.

Broccoli is mostly consumed fresh. The part consumed is thefleshy-stemmed flowering head. Retailers sell such heads in itsentirety. More recently, changes in fresh consumption have taken placein which the broccoli heads are separated into florets. The florets arepackaged and sold as convenience foods, either solely as broccoliflorets or as florets mixed with other fresh vegetables.

Many broccoli varieties grow best on well-drained soils that hold water.In sandy soils, irrigation is important for optimum plant growth and tomaintain proper main curd. Curds develop relative to ambienttemperatures, and in the heat of summer, broccoli curds maturing in Julymay produce flowers and seeds more quickly (four to six days) than thosematuring in the cooler spring and fall periods.

To be considered good quality, broccoli curds should be closed, compact,dark green and tight (no yellow petals showing). A deep green, uniformcurd color is a desirable trait in broccoli. Broccoli heads “green”according to the amount of sunshine reaching the crown of the curds, thecrown being the upper surface of the broccoli curd covered by theflorets. Current commercial ‘heading broccoli’ varieties all have a highleaf canopy that shades at least portions of the curd, particularly atthe margin of the crown, resulting in yellowing around the outerextremities of the harvested broccoli curds, sometimes even causingextensive yellowing of individual florets at the center of the crown.

In a study reviewing harvest practices from 1995 to 1999 in California,broccoli production costs broken down into 29 percent for landpreparation, planting, and growing costs, 45 percent for harvest andpost-harvest costs, 20 percent for cash overhead, 1 percent for intereston operating capital, and 5 percent for non-cash overhead costs. Hence,the labor requirements for harvesting are well over 50% of the totallabor costs for growing broccoli. Since harvesting is the single mostexpensive cultural operation, it is imperative that these costs be keptto a minimum. One trend is to harvest only the main terminal curd,usually by hand. Certain mechanical harvest aids are used, but completemechanical harvesting has not been adopted. Use of modern, more uniformhybrids has enabled growers to complete harvesting in two or, at themost, three manual cuts through the field.

Converting from hand to machine harvesting of broccoli could reducethese labor requirements by a great deal. However, in testing differentcultivars, transplant times, growing techniques and harvest methods, arecent study determined that once over mechanical harvest of broccoliinflorescences, or curds, compared to the graduated traditionalhand-harvest (picking repeatedly 6 to 8 times), results in a yieldreduction on the order of 49% to 60%, depending on the variety. Acombination of hand harvest for the primary curds, followed by amechanical picking of the secondary curds was proposed as reducing yieldlosses, though still on the order of about 23%.

Another issue is the cost of processing broccoli after harvesting.Broccoli curds are often further processed into individual florets,mostly by hand, for freezing or to satisfy market demand for minimallyprocessed fresh vegetable products. The amount of hand labor is verygreat to cut broccoli curds into separate florets, i.e., floretting.Before floretting of the main curd, it must often be first defoliated.In addition to requiring a great deal of hand labor, the floretsseparated individually from the curds are generally not very uniform insize and color, and often require additional trimming and processingbefore packaging. In the UK, the cost of harvesting broccoli in thefield is about $50 per ton. The floretting cost per ton is approximately$120 to $140 ex field. The cost for floretting in a pack house isapproximately $90 per ton.

Floretting broccoli curds for use as minimally processed food isgenerally done at the pack house away from the growing site for hygienicreasons. For this fresh market niche, floret size is preferred in therange of 20 to 80 mm, and with a trimmed square at the cut end. Broccoliis also increasingly being grown and floretted (florets cut from thehead) for frozen products. For the frozen foods market, floret size ispreferred in the range of 20 to 40 mm up to 40 to 65 mm, with the 20 to40 mm being most valuable. Freezers prefer florets that are uniform insize and green color, as more attractive to the consumer.

Green florets are preferred, though a yellow halo is inevitable, giventhe growth habits of current varieties. About 10% pale yellow to darkcream is tolerated on the florets. All present commercial headingbroccoli varieties have curds with very tightly packed florets. Thisresults in individual florets, once separated from the curds, having theappearance of being partially yellow in color around the edges.Additionally, all present commercial broccoli varieties have curds thatare placed deeply within the canopy. As a result of the shading and lackof uniform direct sunlight, curds, as well as the stalk, do no greenuniformly, but show yellow discoloration, especially around theperimeter of the curd.

Therefore, there remains a need in the art for a broccoli plant typeadapted for easier processing by producing a curd with florets uniformlygreen in color.

SUMMARY OF THE INVENTION

The present invention provides a seed of a broccoli plant capable ofproducing a plant comprising a curd having florets, where the florets onthe curd have an average of less than 15% yellowing.

The present invention further provides a seed of a broccoli plantcapable of producing a plant comprising a curd having detached florets,where at least 50% of said florets on the curd do not touch anotherfloret on said curd.

The present invention also provides seed of a broccoli plant capable ofproducing a plant comprising a curd having florets, where the florets onthe curd have an average length of at least about 10 centimeters.

The present invention also provides a container of broccoli seeds wherecurds grown from broccoli plants from greater than 50% of the seeds havedetached florets.

Also provided are containers of broccoli seeds where curds grown fromgreater than 50% of the seeds have detached florets, wherein greaterthan 50% of the florets on the curd do not touch another floret on thesame curd.

The present invention further provides a container of broccoli floretsfrom a broccoli plant having a curd comprising detached florets, wherethe florets have an average of less than 15% yellowing.

The present invention also provides a broccoli plant having a curd withdetached florets.

The present invention also provides a seed of a broccoli plant capableof producing a broccoli plant having a curd with detached florets.

Also provides are parts of a broccoli plant having a curd with detachedflorets, where the curd comprises at least 50% detached florets.

The present invention also provides a method of producing a broccoliseed comprising crossing a first parent broccoli line with a secondbroccoli line, wherein said first parent broccoli line comprisesdetached florets; and obtaining F₁ seed.

The present invention further provides a method of producing a broccoliplant having florets having an average yellowing of less than 15%,comprising crossing a first parent broccoli line with a second broccoliline, where the first parent broccoli line comprises curds havingflorets having an average of less than 15% yellowing; and obtaining F₁seed.

The present invention also provides seed of broccoli line 550478, asample of the seed having been deposited under NCIMB Accession No.41416. The present invention also provides a seed of broccoli line550479, a sample of the seed having been deposited under NCIMB AccessionNo. 41415. The present invention also provides a seed of broccoli line550385, a sample of the seed having been deposited under NCIMB AccessionNo. 41417. The present invention also provides a seed of broccoli line550198, a sample of the seed having been deposited under NCIMB AccessionNo. 41418.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a chart showing the pedigree of the development of thedetached floret lines 550478, 550479, and 550385.

FIG. 2 is a chart showing the pedigree of the development of thedetached floret lines 550475 and 550198.

FIG. 3 is a chart showing the pedigree of the development of thedetached floret line 560465.

FIG. 4 provides a photograph of severed florets from broccoli typeRS1149.

FIG. 5 provides a photograph of a head of an embodiment of a broccoliline of the present invention.

FIG. 6 provides a photograph of severed florets from one embodiment of abroccoli line of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a broccoli plant having a growth typecomprising a main curd that has elongated secondary stems supportingdetached florets. The present invention also provides a broccoli planthaving a curd comprising detached florets that are uniformly green, anddo not show substantial yellowing. In contrast, broccoli florets fromcurrent commercial heading broccoli cultivars are tightly packed into acurd and produce florets, especially interior florets that aresubstantially yellow.

Definitions

Technical or scientific terms used herein shall have the ordinarymeaning accepted by those of skill in the art, unless defineddifferently herein. Descriptions of botanical terms can be found innumerous texts on the subject. See, for example, Hickey, M., and King,C., (2001). Cambridge Illustrated Glossary of Botanical Terms,Cambridge, UK: Cambridge University Press.

The term “broccoli plant” refers to broccoli plants of species Brassicaoleracea L. var italica embracing broccoli varieties, breeding lines,inbred lines, hybrids, and the like.

A “variety” or “cultivar” is used herein in conformity with theInternational Union for the Protection of New Varieties of Plants(“UPOV”) convention and refers to a plant grouping within a singlebotanical taxon of the lowest known rank, which grouping can be definedby the expression of the characteristics resulting from a given genotypeor combination of genotypes, can be distinguished from any other plantgrouping by the expression of at least one of the said characteristicsand is considered as a unit with regard to its suitability for beingpropagated unchanged (stable).

The term “commercial broccoli cultivar” or, “commercial headingbroccoli”, as used herein, refers to currently available broccolicultivars (or varieties), such as Marathon, Decathlon, Triathlon,Heritage, Legacy and Ironman. Such varieties are also referred to as“traditional” varieties.

“Growth type” or “type” refers to one or more morphological (phenotypic)features of a plant, such as overall plant height, leafpresence/absence, leaf size and position, curd height, curd structure,curd size/weight, length of branching, etc.

“Secondary stem” as that term is used herein, means a stalk thatbranches from the main stalk of the broccoli plant above the point ofsevering the harvested head and supporting and forming part ofindividual florets.

As used herein, a “floret” refers to the flower bud cluster includingthat part of the secondary stem supporting the flower bud cluster, whichcollectively make up the curd. The secondary stem branches from the mainstem, and divides further into tertiary, quartenary, etc. stems until atthe end a single filament supports an individual flower bud. The floretprovides a dense cluster of unopened broccoli flower buds, with thefloret having a diameter of 40 to 80 mm, preferably having a diameter of40 to 60 mm. These unopened flower buds are also referred to herein as“beads.”

The term “curd,” as used herein, refers to a collection of florets in asingle floral structure. These florets traditionally form a solid curd.The term “main curd” refers to the curd that forms at the terminus ofthe main stem and excludes secondary curds from side shoots, whichoriginate from the axillary buds of the leaves. The term “head” is alsoused to refer to this floral structure.

The term “semi-detached floret” refers to traditional broccoli plantsshowing some intermediate exsertion of the curd. For broccoli havingsemi-detached florets, the individual florets are not standing alone,but remain close and touching each other. Some exserted head broccolihas the trait of semi-detached florets, for instance SVR 5 is an exampleof a semi-detached floret type. For such lines, the secondary stem isshorter than is the case for detached floret broccoli. SVR 5 wasproduced by crossing PLH2546/PLH33 with BRM53-3921SC, a proprietarySeminis line. Line PLH2546/PLH33, was also used in the development ofthe lines of the present application.

The term “detached florets” refers to a plant growth type with a curd inwhich the secondary stems or stalks are longer than the secondary stalksof commercial broccoli cultivars, on the order of a minimum of 5centimeters in total length measured from the point of branching fromthe main stalk to the tip of the floret. Detached florets also grow insuch a way as to permit a 5-6 cm (diameter) floret to stand alone in thecurd and be separated from the neighbor florets (no touching or at mostde minimis touching of less than a few radians of the floretcircumference). In one aspect, detached florets have less than 10, 9, 8,7, 6, 5, 4, 3, 2, 1 flower buds in contact with a flower bud of anotherfloret. In a preferred aspect, a detached floret has no flower buds incontact with a flower bud on another floret. Preferred florets have alength of 5 to 8 cm. In another aspect, florets have a length of atleast 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 cm. The detached florets grow together from aprimary stalk, forming a more open curd than traditional broccoli, whichpermits easier processing of the florets at harvest for fresh market orfreezing, either by hand or by mechanized means. In one aspect, at least50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the florets on acurd do not contact another floret on the same curd.

“Yellowing” or “discoloration” refers to the presence of yellow flowerbuds in the florets (e.g., generally at the margins) as a result ofshading. A floret having “substantially no yellowing” refers to a florethaving less than 15% yellowing as measured by the percentage of coverageacross the floret having a yellow appearance. A floret having an“absence of yellowing” refers to a floret having less than about 5%yellowing as measured by the percentage of coverage across the florethaving a more yellow than green appearance. In another aspect, floretson a curd have an average of less than 15%, 14%, 13%, 12%, 11%, 10%,7.5%, 5% yellowing as measured by the percentage of coverage across thefloret having a more yellow than green appearance. Yellowing can bemeasured at any time during the development of the florets, at harvest,or post harvest after a period of storage. In a preferred aspect, theyellowing is measured at maturity of the floret at harvest, as measuredby the percentage of coverage across the floret having a more yellowthan green appearance. In a preferred aspect, yellowing is measured asan average yellowing of a population of florets from a curd. In apreferred aspect, a broccoli plant of the present invention comprises acurd having an average yellowing of less than about 15%, 14%, 13%, 12%,11%, 10%, 9%, 8%, 7%, 6%, 5% as measured by the percentage of coverageacross the florets having a more yellow than green appearance.

The term “uniformly green” refers to a curd or a floret havingsubstantially no yellowing. A uniform green floret is substantiallygreen. This color attribute extends to the entire floret, including thepart of the secondary stem supporting the flower bud cluster. Colorclassifications used to describe the florets are based on the colorchart of the Royal Horticultural Society (RHS Color Chart), which is thestandard reference for flower colors and used by specialistsorganizations such as the International Union for the Protection of NewVarieties of Plants (UPOV). Florets of the plants of the invention areclassified in the “green group” and are uniform for the color (forexample 137A/B, 138A/B). Florets of traditional broccoli varieties havesubstantial portions, an average of at least 15% of the individualflorets on a curd, which are classified in the “yellow-green” group (forexample 144B/C, 149D-150D, and 154B/C/D). Individual florets ontraditional broccoli varieties may have as low as 10% yellowing. Thiscolor difference is based on the color of the florets (including stems)when viewed from the side after separation from the curd. The stem ofthe florets of traditional broccoli varieties also classify in the“yellow-green” group (144 B/C and 145 B/C/D) while the stems of theflorets of the present invention classify in the “green” group (137A/B,138A/B/C, and 139D) This description is in accordance with UPOVterminology and the color terminology herein is in accordance with TheRoyal Horticultural Society Colour Chart (R.H.S.C.C.) and the colordescriptions refer to plate numbers in the aforementioned color chart.Color designations, color descriptions and other phenotypic descriptionsmay deviate from the stated values and descriptions depending uponvariation in environmental, seasonal, climatic and cultural conditions.Where a color reference is given these refer to the RHS Colour Chart,The Royal Horticultural Society, London. 2001 Edition.

“Uniformity” refers to a field of plants being uniform in theirphenotypic appearance and development, especially with respect to plantmaturity, curd height and structure, leafless stalk, absence ofyellowing, etc. The harvested plants will, therefore, also be uniform inmaturity, appearance, shelf-life, firmness, etc. Obviously, uniformitydoes not exclude some degree of plant to plant variation, but variationof a uniform crop is minimal. Depending on the trait measured, meanplant to plant variation of a field is preferably less than 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, more preferably less than 1%. Further, themechanical harvesting will enable a uniform (pre-determined) stalklength to be present in the harvested curd.

“Shelf-life” refers to the time period after harvest during which theplants (the curd and/or the stalk) can be stored without quality loss,such as discoloration and loss of firmness. The shelf-life depends onthe genetic make-up of the plant and storage conditions such astemperature, relative humidity, light, etc. In a preferred aspect, atzero degrees Celsius and 95-100% relative humidity the shelf-life ispreferably at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20days, or more.

A “harvested plant” or “harvested broccoli” or “harvested head” refersto the severed curd, comprising part of or the entire main or primarystalk. Preferably, the harvester can be set to cut a specific height (cmdistance) above ground, so that the length of the leafless stalkattached to the harvested head can be predetermined and is uniform forthe harvested crop (the leafless stalk present may for example be 5, 10,15, 20, 25 cm long or more).

Reference to an element by the indefinite article “a” or “an” does notexclude the possibility that more than one of the element is present,unless the context clearly requires that there be one and only one ofthe elements. The indefinite article “a” or “an” thus usually means “atleast one.”

The term “comprising” is to be interpreted as specifying the presence ofthe stated parts, steps or components, but does not exclude the presenceof one or more additional parts, steps or components. A plant comprisinga certain trait may thus comprise additional traits.

Whenever reference to plants according to the invention is made, it isunderstood that also plant parts (cells, tissues, seeds, severed partssuch as curds and/or stalks) are encompassed therein. Also encompassedare progeny of the plants which retain the distinguishingcharacteristics of the parents (especially the uniform green floretcolor and/or detached floret traits), such as seed obtained by selfingor crossing, e.g. hybrid seed, obtained by crossing two inbred parentallines, hybrid plants and plant parts derived therefrom, unless otherwiseindicated.

Traits of Broccoli with Detached Florets

Current commercial heading broccoli has a thick main stem that branchesinto, sturdy secondary stems with an average length of 5 to 6centimeters each supporting a floret.

In one aspect, the present application provides broccoli plants havingdetached florets. In one aspect, the broccoli plants have detachedflorets, such that the slender, sturdy secondary stems each supportingand being part of a floret are substantially longer, or extended, thansecondary stems of current commercial heading broccoli. In one aspect,the secondary stems are about 7, 8, 9, 10 or more centimeters long. Inanother aspect, a curd has detached florets having an average length ofthe secondary stem of about 7, 8, 9, 10, or more centimeters.

In another aspect, the broccoli plants of the present invention havecurds comprising at least six detached florets, and, more preferably, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more florets. In oneaspect, the secondary stems support detached florets that form a mainhead or curd.

In another aspect, the present invention provides a broccoli planthaving a curd in which at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98% or greater of the florets are not touching any otherfloret on the curd. In another aspect, a broccoli plant has a curdhaving florets which have about, or at least about, 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more millimeters between the florets.

The present invention also provides broccoli plants having a curd of auniform green color, ranging from deep green to light green (RHS ColorChart “green group”). In one aspect, the broccoli plants havesubstantially no yellowing, preferably the broccoli plants have a curdhaving an absence of yellowing. While not intending to be limited to anyparticular theory, the detached florets develop a uniform green color assunlight access and penetration is facilitated by the detached floretgrowth habit. In one aspect, substantially no yellowing is seen on thedetached florets because the florets each stand separately from the nextand are exposed to full sunlight across the floret surface. Stems andparts of the florets of traditional broccoli varieties are yellow-greenaccording to the RHS Color Chart.

In another aspect, the broccoli plants of this invention have a curdwith detached florets having a secondary stem length of at least 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 cm. In one aspect, abroccoli plant has detached florets having a secondary stem lengthbetween about 7 cm and about 30 cm, between about 7 cm and about 27.5cm, between about 8 cm and about 30 cm, and between about 9 cm and about25 cm. In a preferred aspect, the length of the secondary stem ismeasured from the tip of the floret to the base of the secondary stem atthe point of attachment to the primary stem. In another aspect, thebroccoli plants of the present invention have detached florets having adiameters of about, or at least about 3, 4, 5, 6, 7, 8, 9 cm. In anotheraspect, the detached florets of the broccoli plants of the presentinvention are between 4 and 10 cm, preferably between 4 and 9 cm, or 4and 8 cm and most preferably between 4 and 6 centimeters in diameter. Inone aspect, a narrower diameter range may be preferred among apopulation of florets for uniformity in packaging. Diameter can bemeasured as a cross section measurement taken through the flower budstructure at the widest point.

In one aspect of the present invention, detached florets are more easilysevered from the curds than is the case for traditional curds of thecurrent compact heading commercial broccoli varieties.

In another aspect, the detached florets are also firm and compact,consisting of closely packed beads or flower buds typical of headingbroccoli. In a preferred aspect, the detached florets of the presentinvention do not have the loose bead cluster growth type typical ofbroccolini or broccoli raab. The firmness of broccoli florets can bedetermined through visual inspection or by measuring the number of beadsper square centimeter of floret surface.

The broccoli plants of the present invention preferably have firmflorets. In one aspect, a firm, or tightly packed, floret has about, orat least about, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 flower buds per squarecm. In one aspect, individual flower buds (beads) range in size between1.50 and 2.25 mm. In one aspect, the buds are not all in one plane orsphere, but at different heights, and are not uniform in size, with budsin the middle, younger florets being smaller than the outer ones. In oneaspect, florets in the apex of the curd of the plant are more tightlyarranged, and a few may lack the fully detached character.

In one aspect, the broccoli plants of the present invention have floretsthat have a weight of about, or at least about, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 grams.Weight of the floret can be measured using any method available. In apreferred aspect, the weight of the floret is measured by weighing thefloret cut to the length equal to the diameter of the floret (the“weight of the square floret”). In one aspect, the florets from abroccoli curd have an average weight of about 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 grams.

In one preferred aspect, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% of the florets on a broccoli curd donot contact another floret on the same broccoli curd. In a preferredaspect, none of the florets on a broccoli curd contact another floret.In one aspect, the broccoli plants of the present invention have curdsthat allow for ease of harvest of the broccoli curds and separation ofthe florets from the curd. In another aspect, the broccoli curds of thepresent invention have detached florets which allow for increasedexposure to sunlight for each of the florets on the curd. In one aspect,the detached florets receive 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%sunlight over the surface area of each floret on the broccoli curd. Inone aspect, where the florets are not touching, uniform coloring bysunlight results.

In another aspect of the present invention the curd of the broccoliplant of the invention is also exserted above the leaf canopy. In oneaspect, the curd exsertion contributes to even greater sunlight exposureto the detached florets.

As used herein, “raised head” or “exserted head” refers to a plantgrowth type, wherein the curd develops above the leaf canopy of theplant. In one aspect, the crown of the curd is raised by at least about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or more centimeters above the leaf canopy. The “crown”refers to the uppermost part of the curd, i.e., top of the curd coveredby the packed inflorescences. “Below the head” refers to the area belowthe curd, i.e., below the attachment of the substantial portion of theflorets to the stem and above the point of severing the head at harvest.Such broccoli plants are described in the U.S. patent application Ser.No. 10/850,077 entitled “Broccoli Type Adapted For Ease of Harvest,”Publication No. 20050262594, the contents of which are incorporated intheir entirety by reference.

Exserted curds are at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24 centimeters higher than the top of the canopy, and inpreferred embodiments at least 25 centimeters higher than the top of thecanopy. In addition, exserted broccoli plant types may have asubstantial absence of leaves and leaflets along the stalk immediatelybelow the curd. This also allows for easier harvesting, as curds do notrequire defoliation after harvesting. In one embodiment, within 25centimeters below the crown of the curd, the plant producessubstantially no leaves or petioles having a surface area greater thanabout 30 square centimeters, preferably greater than about 20 squarecentimeters. In a particularly preferred embodiment, substantially noleaves or petioles are produced within 25 centimeters below the crown.

The present invention also provides a seed of a broccoli plant capableof producing a broccoli plant in which curds obtained from broccoliplants grown for the seed have detached florets.

In another aspect, the present invention also provides for a plant grownfrom the seed of a broccoli plant in which curds obtained from broccoliplants grown for the seed have a detached floret trait, as well as plantparts and tissue cultures from such plants.

The present invention also provides a container of broccoli seeds inwhich curds obtained from broccoli plants grown from greater than 50% ofthe seeds have a detached floret trait. In another aspect, curdsobtained from broccoli plants grown from greater than 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% of the broccoli seeds in the container havea detached floret trait.

The container of broccoli seeds may contain any number, weight or volumeof seeds. For example, a container can contain at least, or greaterthan, about 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more seeds.Alternatively, the container can contain at least, or greater than,about 1 ounce, 5 ounces, 10, ounces, 1 pound, 2 pounds, 3 pounds, 4pounds, 5 pounds or more seeds.

Containers of broccoli seeds may be any container available in the art.By way of non-limiting example, a container may be a box, a bag, apacket, a pouch, a tape roll, a pail, a foil, or a tube.

In another aspect, the seeds contained in the containers of broccoliseeds can be treated or untreated broccoli seeds. In one aspect, theseeds can be treated to improve germination, for example, by priming theseeds, or by disinfection to protect against seed-born pathogens. Inanother aspect, seeds can be coated with any available coating toimprove, for example, plantability, seed emergence, and protectionagainst seed-born pathogens. Seed coating can be any form of seedcoating including, but not limited to pelleting, film coating, andencrustments.

In another aspect, the present invention also provides a container ofbroccoli florets in which greater than 50% of the florets are obtainedfrom a broccoli plant having detached florets. In another aspect,greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the floretsin the container are obtained from a broccoli plant having detachedflorets.

In another aspect, the present invention provides a container ofbroccoli florets in which greater than 50% of the florets havesubstantially no yellowing. In a preferred aspect, greater than 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the florets havesubstantially no yellowing. In another aspect, greater than 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the florets in thecontainer have an absence of yellowing.

The container of florets may contain any number, weight or volume offlorets. For example, a container can contain at least, or greater than,about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 100 florets.Alternatively, the container can contain at least, or greater than,about 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more florets.

Containers of florets may be any container available in the art. By wayof nonlimiting example, a container may be a box, a flat, a clamshell, abag, a packet, or a bunch. A container of florets of the presentinvention may be found in any location, including, but not limited to awarehouse, a distributor, a wholesaler, or a retail market, such as agrocery store.

Breeding and Development of Inbreds and Hybrids with the Detached FloretGrowth Type

Any broccoli plant having a suitable floret type can be used inconjunction with the present invention. Broccoli plants with suitablefloret type can be used in the methods of the present invention. In apreferred aspect, the source of a detached floret type is an inbredbroccoli plant. In an aspect, suitable detached floret broccoli plantsmay be produced by breeding with the following sources: DH MRE-7, DHMRD-1, GM-1.6, B319, DH E-47, EC-2, DH GV-37, SH-2, OSU-102, OSU-111,MRD-2, HC-1, BRM56-3905, BRM53-3921, DC3EC6, 1032-1104 and 2151×T&A. Inone aspect, a detached floret source broccoli plant can be identified bydetermining the floret type of a source broccoli plant, for example assemi-detached floret type or an exserted head type. In another aspect, adetached floret source broccoli plant may be crossed with any inbred orelite broccoli line having desired properties.

In one aspect, a detached floret-plant type can be identified in theprogeny of certain crosses after microspore culture. Microspore culturecan be used in exploiting recessive characteristics, and can alsoprovide a method of producing genetically stable homozygous lines whenfixed by chromosome doubling. Microspore culture is a breeding tool thatis well known in the art, for example in U.S. Pat. No. 6,200,808, theentirety of which is incorporated herein by reference. In one aspect,continuous selection for raised head broccoli plant types can be used toobtain broccoli plant types with curds of a variable structure. In oneaspect, such methods produce erect and tall plant types, supporting acompact curd with densely packed compact florets. Such methods can alsoproduce shorter and less erect plant types, but with the secondarybranches of the florets extending from the main stem under a largerangle (more horizontal). However, because of the shortness of thesecondary stems, the florets touch each other. Such growth types can beclassified as plants having a semi-detached curd. In one aspect, thebroccoli plants of the present invention can be produced using crossesbetween the semi-detached broccoli plants and erect plants with anexserted curd, in combination with microspore culture.

The broccoli plants of the present invention can be homozygous orheterozygous. Plants that have been self or sib-pollinated once or twicefollowed by self or sib pollination and selected for type over manygenerations may retain some genotypic heterozygosity, but becomephenotypically uniform. A cross between two such heterozygous buthomogeneous parents produces a phenotypically uniform population ofhybrid plants that are heterozygous for many gene loci. The developmentof such parent lines generally requires at least about 5 to 7generations of selfing and/or sib mating. Two such parent lines can thenbe crossed to develop improved F₁ hybrids. Hybrids can then be screenedand evaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, can bemeasured. In another aspect, dihaploid plants are developed and theseplants are genotypically uniform. Two such plants can be crossed, or aconventionally produced parent line described above can be crossed witha dihaploid, producing F₁ hybrids that are evaluated as described above.

The present invention also provides progeny of broccoli plants havingdetached florets. As used herein, the progeny include not only, withoutlimitation, the products of any cross (be it a backcross or otherwise)between two plants, but all progeny whose pedigree traces back to theoriginal cross. Specifically, without limitation, such progeny includeplants that have 50%, 25%, 12.5% or less genetic material derived fromone of the two originally crossed plants. As used herein, a second plantis derived from a first plant if the second plant's pedigree includesthe first plant.

Broccoli plants generated using a method of the present invention can bepart of or generated from a breeding program. The choice of breedingmethod depends on the mode of plant reproduction, the heritability ofthe trait(s) being improved, and the type of cultivar used commercially(e.g., F₁ hybrid cultivar, pureline cultivar, etc). Selected,non-limiting approaches, for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingany method available, such as, marker assisted selection of the progenyof any cross. It is further understood that any commercial andnon-commercial cultivars can be utilized in a breeding program. Factorssuch as, for example, emergence vigor, vegetative vigor, stresstolerance, disease resistance, branching, flowering, seed set, seeddensity, curd size, exsertion etc. will generally dictate the choice.

The present invention provides processes of preparing novel broccoliplants and broccoli plants produced by such processes. In accordancewith such a process, a first parent broccoli plant may be crossed with asecond parent broccoli plant wherein at least one of the first andsecond broccoli plants is a parent line or dihaploid line or detachedfloret broccoli plant as described herein. One application of theprocess is in the production of F₁ hybrid plants. Another importantaspect of this process is that the process can be used for thedevelopment of novel parent, dihaploid or inbred lines. For example, adetached floret broccoli plant as described herein could be crossed toany second plant, and the resulting hybrid progeny each selfed and/orsibbed for about 5 to 7 or more generations, thereby providing a largenumber of distinct, parent lines. These parent lines could then becrossed with other lines and the resulting hybrid progeny analyzed forbeneficial characteristics. In this way, novel lines conferringdesirable characteristics could be identified.

Broccoli plants (Brassica oleracea L. var. italica) can be crossed byeither natural or mechanical techniques. Mechanical pollination can beeffected either by controlling the types of pollen that can betransferred onto the stigma or by pollinating by hand.

In another aspect, the present invention provides a method of producinga broccoli plant having detached florets comprising: (a) crossing afirst broccoli line with a second broccoli line to form a segregatingpopulation, where the first broccoli line has detached or semi-detachedflorets; (b) screening the population for detached florets; and (c)selecting one or more members of the population having detached florets.

In another aspect, the present invention provides a method ofintrogressing a detached floret trait into a broccoli plant comprising:(a) crossing at least a first broccoli line having detached orsemi-detached florets with a second broccoli line to form a segregatingpopulation; (b) screening the population for detached florets; and (c)selecting at least one member of the population having detached florets.

Parental plants are typically planted in pollinating proximity to eachother by planting the parental plants in alternating rows, in blocks orin any other convenient planting pattern. Where the parental plantsdiffer in timing of sexual maturity, it may be desired to plant theslower maturing plant first, thereby ensuring the availability of pollenfrom the male parent during the time at which the stigmas on the femaleparent are receptive to pollen. Plants of both parental parents arecultivated and allowed to grow until the time of flowering.Advantageously, during this growth stage, plants are in general treatedwith fertilizer and/or other agricultural chemicals as consideredappropriate by the grower.

Alternatively, in another aspect of the invention, both first and secondparent broccoli plants can be a detached floret broccoli plant asdescribed herein. Thus, any broccoli plant produced using a detachedfloret broccoli plant as described herein forms a part of the invention.As used herein, crossing can mean selling, sibbing, backcrossing,crossing to another or the same parent line, crossing to populations,and the like. All broccoli plants produced using a detached floretbroccoli plant as described herein as a parent are, therefore, withinthe scope of this invention.

In another aspect, the present invention provides a hybrid broccoliplant having detached florets. In another aspect, the present inventionprovides seed of a hybrid broccoli plant having detached florets. Anytime a detached floret broccoli plant as described herein is crossedwith another, different, broccoli parent line, a first generation (F₁)broccoli hybrid plant is produced. As such, an F₁ hybrid broccoli plantcan be produced by crossing a detached floret broccoli plant, forexample, as described herein with any second parent broccoli plant.Essentially any other broccoli plant can be used to produce a hybridbroccoli plant having a detached floret broccoli plant as describedherein as one parent. All that is required is that, at a minimum, oneplant be female fertile and the second plant be male fertile.

A single cross hybrid broccoli variety is the cross of two parent lines,each of which has a genotype which complements the genotype of theother. Typically, F₁ hybrids are more vigorous than their parents. Thishybrid vigor, or heterosis, is manifested in many polygenic traits,including improved yields, better roots, better uniformity and betterinsect and disease resistance. In the development of hybrids only the F₁hybrid plants are typically sought. An F₁ single cross hybrid isproduced when two parent plants are crossed. A double cross hybrid isproduced from four parent plants crossed in pairs (A×B and C×D) and thenthe two F₁ hybrids are crossed again (A×B)×(C×D). A three-way cross isproduced from three parent plants crossed as (A×B)×C.

In one aspect, any of the broccoli varieties known to those of skill inthe art can be crossed with a detached floret broccoli line of thepresent invention to produce a hybrid plant. In a preferred aspect, suchbroccoli varieties include, but are not limited to Marathon, Decathalon,Heritage, Legacy, and Ironman.

When a detached floret broccoli plant as described herein is crossedwith another parent plant to yield a hybrid, it can serve as either thematernal or paternal plant. For many crosses, the outcome is the sameregardless of the assigned sex of the parental plants. Depending on theseed production characteristics relative to a second parent in a hybridcross, it may be desired to use one of the parental plants as the maleor female parent. Therefore, a decision to use one parent plant as amale or female may be made based on any such characteristics as is wellknown to those of skill in the art.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing a detached floret broccoli plant as describedherein followed by multiple generations of breeding according to suchwell known methods. New varieties may be created by crossing a detachedfloret broccoli plant as described herein with any second plant. Inselecting such a second plant to cross for the purpose of developingnovel parent lines, it may be desired to choose those plants whicheither themselves exhibit one or more selected desirable characteristicsor which exhibit the desired characteristic(s) when in hybridcombination. Examples of potentially desired characteristics includegreater yield, resistance to insecticides, herbicides, pests, anddisease, tolerance to heat and drought, reduced time to crop maturity,better agronomic quality, higher nutritional value, and uniformity ingermination times, growth rate, maturity, and root size.

Once initial crosses have been made with a detached floret broccoli lineof the present invention, selfing and/or sibbing take place to producenew parent lines. The development of parent lines requires manipulationby human breeders. A combination of self pollination and sib pollinationis essential to develop a new parent line that is genotypically stableand phenotypically uniform. The reason for the breeder to create parentlines is to develop homogeneous populations in an outcrossing speciesthat are phenotypically uniform and can be utilized to produce F₁hybrids.

The pedigree breeding method involves crossing two genotypes. Eachgenotype can have one or more desirable characteristics lacking in theother; or, each genotype can complement the other. If the two originalparental genotypes do not provide all of the desired characteristics,other genotypes can be included in the breeding population. Superiorplants that are the products of these crosses are selfed or sibbed andselected in successive generations. Each succeeding generation becomesmore genetically homogeneous and phenotypically uniform as a result ofself- or sib-pollination and selection. Typically, this method ofbreeding involves five or more generations of selfing or sibbing andselection. After at least five generations, the resulting parent linehas a stable allelic frequency at each locus and is phenotypicallyuniform.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. A genetic locus conferring the traits may ormay not be transgenic. Examples of such traits known to those of skillin the art include, but are not limited to, male sterility, herbicideresistance, resistance for bacterial, fungal, or viral disease, insectresistance, male fertility and enhanced nutritional quality. These genesare generally inherited through the nucleus, but may be inheritedthrough the cytoplasm. Some known exceptions to this are genes for malesterility, some of which are inherited cytoplasmically, but still act asa single locus trait.

Direct selection may be applied where a genetic locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide resistancecharacteristic, and only those plants which have the herbicideresistance gene are used in the subsequent backcross. This process isthen repeated for all additional backcross generations.

Many useful traits are those which are introduced by genetictransformation techniques. Methods for the genetic transformation ofbroccoli are known to those of skill in the art. For example, methodswhich have been described for the genetic transformation of broccoli mayinclude electroporation, electrotransformation, microprojectilebombardment, Agrobacterium-mediated transformation, direct DNA uptaketransformation of protoplasts and silicon carbide fiber-mediatedtransformation. See. e.g., Khachatourians, G., et al., eds., TransgenicPlants and Crops, Marcel Dekker, Inc. (2002) and Fruit and VegetableBiotechnology (Victoriano Valpuesta ed. Woodhead Publ. 2002).

It is understood to those of skill in the art that a transgene need notbe directly transformed into a plant, as techniques for the productionof stably transformed broccoli plants that pass single loci to progenyby Mendelian inheritance is well known in the art. Such loci maytherefore be passed from parent plant to progeny plants by standardplant breeding techniques that are well known in the art. Examples oftraits that may be introduced into a broccoli plant according to theinvention include, for example, male sterility, herbicide resistance,disease resistance, insect resistance, and enhanced nutritional quality.

PCR and Southern hybridization are two examples of molecular techniquesthat can be used for confirmation of the presence of a given locus andthus conversion of that locus.

The detached floret trait can be any genotype, including but not limitedto dominant, recessive, or a quantitative trait. In a preferred aspect,the inheritance of the detached floret broccoli trait recessive. In oneaspect, the detached floret broccoli type is homozygous or heterozygous.In one aspect, microspore culture can be used to develop a homozygousbreeding line. Inbred lines are also important for the development ofdetached floret hybrid plants, where both parents need to have thedetached floret trait.

Broccoli plants of the detached floret growth type, deposited with theNCIMB as described below, or derivatives there from which retain thegrowth types of these plants, can be used as parental lines. Thedetached floret trait may be transferred to other broccoli plants byusing conventional breeding techniques and selecting progeny that retainthe growth type of the parents, i.e. having secondary stems supportingdetached florets with no substantial yellowing around their margins.

Also provided is a method of producing hybrid broccoli plants which havedetached florets. This method involves crossing two (preferablydi-haploid) plants according to the invention and harvesting the hybridseeds. The seeds obtained will, when grown, show the novel growth type,in particular the extended and separated secondary stem stalk anddetached florets, as defined. In one embodiment the method comprises thesteps of planting rows of male and female parents of inbred plantsaccording to the invention in a field, growing the plants untilflowering and seed set has occurred, and harvesting the hybrid seed fromthe row of female parents. The method preferably prevents anyself-pollination of the female parents from occurring in order to obtain100% pure hybrid seeds (see below).

Seed Production

For large scale hybrid seed production, different systems of crosspollination, based on self-incompatibility, or, alternatively,cytoplasmic male sterility (CMS), can be used. These techniques are wellknown in the art. Large scale increase of the hybrid parents (inbredlines) is done by self-pollination, where necessary facilitated byincreasing the concentration of CO₂ to overcome theself-incompatibility, or bud pollination using hand labor. Such largescale increase of inbred lines is most commonly done in a greenhouse orplastic house. This practice of parent line seed production leads togood quality seed and disease control. Inbred broccoli plants accordingto the invention include for example broccoli plants of lines 550478,550479, 550385, 550475, 550198, and 560465, and seeds or derivativesthereof. These lines can by increased by bud pollination or convertedinto a CMS cytoplasm. These methods are well known to a person skilledin the art.

The commercial hybrid seed is produced in the open field byinter-planting rows of the seed (female) parent and the pollinator(male) parent, where self-incompatibility or CMS of the seed parentprevents self pollination and ensures the harvesting of hybrid F1 seed,in methods well know in the art. The method for producing hybrid seedaccording to the invention thus involves in one embodiment the growingof rows of male and female parents having the novel growth type and theharvesting of the hybrid seeds from the female parent rows.

For broccoli hybrid seed production, the modern system uses CMS that wasintrogressed into Brassica oleracea L. from radish. Studies on the newmale sterility in Japanese radish, with special reference to theutilization of this sterility towards practical raising of hybrid seed.

Thus, also provided are inbred broccoli plants according to theinvention which are male sterile and are suitable for being used as afemale parent in hybrid seed production. In one embodiment the inbredbroccoli plants are male sterile due to cytoplasmic male sterility, e.g.the Ogura CMS. Such plants can be made as known in the art. In anotherembodiment the plant according to the invention is genetically malesterile or male sterile due to one or more transgenes conferring malesterility being integrated into their genome, as for example describedin EP 0 344 029; U.S. Pat. Nos. 6,509,516; 5,254,802; or in U.S. Pat.No. 5,789,566, the contents of all of which are incorporated in theirentirety by reference

Seed from various lines of inbred broccoli with the detached floretgrowth type have been deposited with NCIMB, as described below. Thisinvention encompasses, plants and plant parts produced by growing suchseeds, including pollen and an ovule from such plants.

EXAMPLES Example 1: Development of Plant with Curds Having DetachedFlorets

Both proprietary and public research lines were available having anexserted curd or raised head (RH) trait. For instance, the Oregon StateUniversity (OSU) broccoli breeding program had lines with a moderateexserted head, and several accessions were obtained in the 1980's fromthe OSU breeding program. Some of these lines were designated as OSU-102and OSU-111. These accessions produced poor head size, poor headquality, generally, and leaves on the stem just below the head whichrendered such lines unsuitable as parents for commercially viablehybrids. Selection for better raised head (referred to as ‘RH’) traitsand higher internode lengths consistently led to lower head weights.Eventually after multiple rounds of breeding and selection, lines weredeveloped with the RH trait, and compact heavy heads of good quality.Proprietary accessions selected at the start of the breeding projectwere designated DH MRE-7, DH MRD1-1, GM-1.6, B19, DH E-47, EC-2, DHGV-37, SH-2, OSU-102, OSU-111, MRD-2, HC-1, BRM56-3905, BRM53-3921,DC3EC6, 1032-1104 and 2151×T&A. All of these lines were elite parentlines developed in the Seminis breeding program, that were used for theproduction of commercial hybrids as long ago as the 1970s. These lineswere chosen at least partly to compensate for the defects observed ofthe horticultural characteristics of the OSU lines.

More specifically, the proprietary lines had very good general combiningability, resistance to disease, particularly to downy mildew(Peronospora parasitica), already showed reasonably good RH traits, goodhead height and head-height uniformity, as well as resistance tobacterial soft rot (Erwinia and Pseudomonas bacteria). The pedigree forlines 550478, 550198, 550479 and 550385 are provided in FIG. 1. Thepedigree for line 550475 is provided in FIG. 2 and for line 560465 isprovided in FIG. 3.

The designation DH designates double haploid, and indicates that theselines have been developed through microspore culture, followed bychromosome doubling.

All genetic materials or lines have been developed after many years ofcrossing and selection. Details of this crossing and selection programare described in FIGS. 1, 2 and 3. Progeny plants (F1) of each crosswere selected for their phenotypic appearance for head exsertion incombination with favorable horticultural characteristics for all otherimportant horticultural and head traits. The selected plants from thebest families were crossed again with other selected plants from otherfamilies. Occasionally, between two crossing cycles selected plants wereselfed for one or two generations (F2, F3) to obtain better uniformityof the lines.

The best plants of these lines were crossed again. This breedingprocedure is known as the modified family selection, as is described instandard text books of plant breeding, e.g., Allard, R W, Principles ofPlant Breeding (1960) New York, N.Y., Wiley, pp 485; Simnmonds, N W,Principles of Crop Improvement (1979), London, UK, Longman, pp 408;Sneep, J. et al., (1979) Plant breeding perspectives, PUDOC Wageningen,The Netherlands; Mark J. Basset, (ed), Breeding of Vegetable Crops,(1986) AVI Publ. Comp. Westport, U.S.A.; Fehr, W R, et al., Principlesof Cultivar Development—Theory and Technique (1987) MacMillan, New York,N.Y.

In the course of the selection program several lines showing favorablecharacteristics were selected which were designated as PLH, andassociated with a sequence number.

Crossing of lines with curds showing semi-detached florets(BRM56-3905/BRM53-5913) with lines having fully exserted heads(PLH2546/PLH33) led to the development of line 520166(PLH2546/PLH33/BRM56-3905/BRM53-3913). The pedigree is provided in FIG.1.

FIG. 2 shows the pedigree for line 521163. Line 521163, originated froma cross between line PLH42/1032/BRM56-3905/BRM53-3913 (semi-detached)and PLH2546!plh33/brm56-3905.brm53-3913 with fully exserted head. Thisled to the selection of a line designated 521163, which similarly toline 520166 shows branched curds with detached florets.

Example 2: Microspore Culture

Lines 520166 and 521163 were used as the starting material for themicrospore culture. Anthers from heterozygous donor plants grown under a16 h photoperiod at 10-12° C. are used as experimental material. Flowerbuds of different sizes are harvested and isolated, anthers weresquashed and stained with DAPI to determine the stage of meioticdevelopment. Flower buds containing anthers with pollen in lateuni-nucleate to early bi-nucleate stage are selected and sterilized byspraying with 96% ethanol, followed by immersion in a 2.6% solution ofsodium hypochlorite for 5 min and rinsed three times with sterilizeddistilled water.

The sterilized buds are prepared in B5 liquid medium and buds aresquashed as described in Fan, Z., Armstrong, K. C. and Keller, W. A.,Development of microspores in vivo and in vitro in Brassica napus L.,Protoplasma 147, 191-199 (1988). The resulting squashed buds are thenfiltered through two nylon filters (48 μm, 63 μm Ø) and centrifuged (100g, 8° C., 4 min), and the pellet is washed 3 times in 27 ml B5 liquidmedium. A small amount of charcoal is added and the pellet re-suspendedin a liquid culture medium as described in Lichter, R. Z., Antherculture of Brassica napus in a liquid culture medium, Planzenphysiol.103, 229-237 (1981) and in Lichter R. Z., Induction of haploid plantsfrom isolated pollen of Brassica napus, Planzenphysiol. 105, 427-437(1982) with a final density of 1 flower bud per ml of this medium. Themicrospore suspension cultures are incubated in the dark at 35° C. for24 h and then grown in the dark at 25° C.

After 3 to 6 weeks embryos appear and are transferred to a plantregeneration medium, as described by in Keller, W. A. and Armstrong, K.C., Embryogenesis and plant regeneration in Brassica napus anthercultures, Can. J. Bot. 55, 1383-1388 (1977) incubated in continuouslight at 8° C. for 1 week. Subsequently, the embryos were transferred toconditions of 16 h photoperiod of indirect light at 25° C. After 4 weeksplant-like structures with meristems were transferred to a rootingmedium (4.44 g/l MS basic salts including vitamins without myo-inositol(purchased from Duchefa as MS Royal Sluis), 100 mg/l myo-inositol(purchased from BDH), 400 μg/l Thiamine HCl (Duchefa), 200 μg/l IBA(Duchefa), 30 μg/l sucrose (Duchefa), 10 μg/l plant agar (Duchefa), pHadjusted to 5.8) and grown under a 16 h photoperiod at 20° C.Regenerated plantlets are transferred on fresh rooting medium once every4 weeks.

This procedure is continued until plants are delivered to the breederfor further selection and breeding. Plants of unknown ploidy level areprovided to the breeder. The breeder selects those plants that arephenotypically desirable, fertile and produce identical fully homozygousprogeny.

Inbred lines are tested and those that give the best hybrid performanceare identified and selected. Hybrid seed can be produced indefinitely,as long as the homogeneity and the homozygosity of the inbred parents ismaintained.

Heterozygous lines 520166 (PLH25461PLH33/BRM56-3905/BRM53-3913) and521163((PLH42/1032/BRM56-3905/BRM53-3913×PLH2546/PLH33/BRM56-3905/BRM53-3913)were used as the starting material for the microspore experiment.Broccoli lines 550478, 550479, 550198, and 550385 were obtained from themicrospore culture of 520166. Broccoli line 550475 was obtained frommicrospore culture of 521163. These lines were selected on the basis ofthe detached and uniformly green florets combined with desirablehorticultural traits.

Seed samples obtained from representative broccoli lines having detachedflorets were deposited under NCIMB accession numbers as follows: Seedsof broccoli line 550479 were deposited under NCIMB Accession No. 41415;seeds of broccoli line 550478 were deposited under NCIMB Accession No.41416; and seeds of broccoli line 550385 were deposited under NCIMBAccession No. 41417. Seeds of broccoli line 550198 were deposited underNCIMB Accession No. 41418.

Example 3: Additional Inbred Broccoli Lines

Additional inbred broccoli lines are developed from crosses usingbroccoli line 520166, described above. The pedigree of broccoli line560465 is provided in FIG. 3.

Measurements are taken from a commercial crown broccoli hybrid (RS1149,or Tinman), a crown broccoli hybrid line, 550118, and the two detachedfloret lines, 560465 and 550475. FIG. 4 provides a photograph of severalouter florets of commercial broccoli hybrid RS1149, and FIGS. 5 and 6provide photographs of an intact head, and several severed florets,respectively, of broccoli line 550465. The following data are collectedfrom the broccoli plants and are provided in Table 1 below. The minimumdistance of each floret to the nearest floret in the curd (inmillimeters); the widest diameter of the floret (in millimeters); thetotal length of the floret from the crown to the attachment of thefloret to the primary stem (in millimeters); the quality rating of eachfloret (on a scale of 1 to 5; 1 being very poor, 2 being poor, 3 beingmedium, 4 being good, and 5 being very good); the color of the top ofthe floret (using the color chart from the Royal Horticultural Society);the color of the shoulder of the floret (also using the color chart fromthe RHS); the percent yellow of the floret (percent shoulder of thetotal floret); the color of the stem of the floret (based on the RHScolor chart); and the weight of the square floret (the length of thefloret cut to the same size as the diameter of the floret).

TABLE 1 MIN. FLORET % WEIGHT DISTANCE DIAM- TOTAL QUALITY FLORET COLORSHOUL- FLORET SQUARE FIELD PLANT FLO- TO NEAREST ETER LENGTH RATINGCOLOR SHOUL- DER/ COLOR FLORET # MNR # RET# FLORET (mm) (mm) (mm) (1-5)TOP DER TOTAL STEM (gr) 232 RS1149 1 1 3 40 70 4 N138B 154C X 145A 7 2 065 75 4 N138B 154C X 145A 24 3 0 60 60 3 N138B 154C X 145B 18 4 0 75 654 N138B 154C X 145B 37 5 0 70 60 4 N138B 154C X 145C 32 6 0 80 55 4N138B 154C X 145C 33 7 0 65 55 3.5 N138B 154C X 145D 23 8 0 60 50 4N138B 154C X 145D 26 9 0 40 45 4 N138B 154C X 145D 12 10 0 40 40 3 N138B154C X 145D 9 1037 550118 1 1 0 50 60 3 N138B 154C 25 145B 14 2 0 60 504 N138B 154C 20 145B 16 3 0 60 45 4 N138B 154D 25 145B 18 4 0 70 55 4N138B 154D 15 145B 29 5 0 60 50 4 N138B 154D 15 145B 22 6 0 60 50 4N138B 154C 10 145B 21 7 0 50 45 4 N138B 154D 15 145C 14 8 0 50 40 3.5N138B 154D 15 145C 13 9 0 45 40 3 N138B 154D 20 145D 9 10 0 40 40 3N138B 154D 15 145D 8 2 1 0 60 60 4 N138B 154B 15 145B 21 2 0 60 50 4N138B 154C 15 145B 21 3 0 55 50 4 N138B 154B 15 145B 14 4 0 65 50 4N138B 154D 15 145C 19 5 0 65 50 4 N138B 154D 10 145C 25 6 0 50 45 4N138B 154D 10 145D 13 7 0 55 45 3 N138B 154D 10 145D 15 8 0 50 40 3.5N138B 154D 10 145D 12 9 0 45 35 3 N138B 154D 20 145D 9 10 0 40 35 3N138B 154D 15 145D 8 1037 AV 0 54.5 46.75 3.65 N138B 154B-D   15.5145B-D 16.05 1300 560465 1 1 10 50 220 4 N138C NA NA N138C 17 2 12 60200 4 N138C NA NA N138C 20 3 1 55 180 4 N138C NA NA N138C 18 4 30 60 2104 N138C NA NA N138C 17 5 3 60 180 3 N138C NA NA N138C 15 6 3 45 120 3N138C NA NA N138C 18 7 10 50 140 4 N138C NA NA N138C 12 8 3 50 125 3N138C NA NA N138C 9 9 0 40 100 2 N138C NA NA N138C 6 10 0 25 80 2 N138CNA NA N138C 3 2 1 1 35 260 4 N138C NA NA N138C 6 2 20 55 200 4 N138C NANA N138C 17 3 10 50 170 4 N138C NA NA N138C 13 4 14 50 155 4 N138C NA NAN138C 11 5 15 60 125 4 N138C NA NA N138C 12 6 20 45 120 4 N138C NA NAN138C 9 7 14 35 100 4 N138C NA NA N138C 6 8 1 35 75 3 N138C NA NA N138C3 1300 AV 8.35 43 138 3.2 N138C NA NA N138C 11.563 1305 550475 1 1 10 40230 2 139C NA NA 139D 6 2 20 75 170 4 139C NA NA 139D 25 3 2 50 150 3139C NA NA 139D 9 4 5 40 115 4 139C NA NA 139D 7 5 4 55 145 4 139C NA NA139D 20 6 5 50 160 3.5 139C NA NA 139D 14 7 5 45 145 3 139C NA NA 139D 78 5 55 110 4 139C NA NA 139D 13 9 2 45 115 3.5 139C NA NA 139D 8 10 1 4090 3.5 139C NA NA 139D 6 2 1 3 45 280 3 139C NA NA 139D 8 2 13 40 245 2139C NA NA 139D 7 3 5 55 215 3 139C NA NA 139D 15 4 15 55 180 3 139C NANA 139D 13 5 11 60 190 4 139C NA NA 139D 20 6 16 60 175 2 139C NA NA139D 4 7 1 30 115 4 139C NA NA 139D 16 8 6 45 125 4 139C NA NA 139D 9 91 40 111 4 139C NA NA 139D 8 10 3 35 85 3 139C NA NA 139D 3 1305 AV 6.6548 157.6 3.325 139C NA NA 139D 10.9

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

What is claimed is:
 1. A seed of a broccoli plant capable of producing aplant comprising a curd having florets, wherein the traits of saidflorets on said curd comprise: an average of more than 85% of the flowerbuds on said florets having a color score in the green range on theRoyal Horticultural Society color chart selected from the groupconsisting of 137A, 137B, 138A, and 138B, and at least 50% of saidflorets on said curd are not touching another floret on said curd; andwherein said traits are obtainable from a broccoli line selected fromthe group consisting of 550478, a representative sample of seed of saidline having been deposited with the NCIMB under NCIMB Accession No.41416; 550479, a representative sample of seed of said line having beendeposited with the NCIMB under NCIMB Accession No. 41415; 550385, arepresentative sample of seed of said line having been deposited withthe NCIMB under NCIMB Accession No. 41417; and 550198, a representativesample of seed of said line having been deposited with the NCIMB underNCIMB Accession No.
 41418. 2. The seed of claim 1, wherein said floretscomprise stems having a color score in the green range on the RHS colorchart selected from the group consisting of 137A, 137B, 138A, 138B,138C, and 139D.
 3. The seed of claim 1, wherein said curd has at least 8florets.
 4. The seed of claim 1, wherein at least 60% of said florets onsaid curd do not touch another floret on the same curd.
 5. The seed ofclaim 1, wherein said florets have an average of less than 5% yellowingaround the edges as measured by the percentage of coverage across thefloret having a more yellow than green appearance at maturity of thefloret at harvest.
 6. The seed claim 1, wherein said florets have anaverage secondary stem length of between 7 centimeters (cm) and 30 cm.7. The seed of claim 1, wherein said florets have an average weight ofat least 2 grams.
 8. The seed of claim 1, wherein said florets arebetween 4 cm and 10 cm in diameter.
 9. The seed of claim 1, wherein saidcurd of said broccoli plant comprises six or more florets.
 10. The seedof claim 1, wherein said seed is a hybrid seed, produced by a cross of abroccoli plant deposited under NCIMB Accession No. 41416, 41415, 41417or 41418 as a male parent and a second broccoli plant as a femaleparent, wherein said hybrid plant comprises a curd having florets,wherein said florets on said curd have an average of more than 85% ofthe flower buds on said florets having a color score in the green rangeon the Royal Horticultural Society color chart selected from the groupconsisting of 137A, 137B, 138A, and 138B, and said curd has a traitselected from the group consisting of florets with less than 10 flowerbuds in contact with a flower bud of another floret in the same curd; asecondary stem of a length of between 7 cm and 30 cm; and combinationsthereof.
 11. A container of one or more of the broccoli seeds ofclaim
 1. 12. A broccoli plant grown from the seed of claim
 1. 13. Thebroccoli plant of claim 12, wherein said florets have an average weightof at least 2 grams.
 14. The broccoli plant of claim 12, wherein saidflorets are between 4 cm and 10 cm in diameter.
 15. The broccoli plantof claim 12, wherein said curd comprises at least six or more florets.16. The broccoli plant of claim 12, wherein said secondary stem has anaverage length of between 7 cm and 30 cm.
 17. A part of the broccoliplant of claim
 12. 18. The part of a broccoli plant of claim 17, whereinsaid part is selected from the group consisting of seed, endosperm,ovule, and pollen.